Epstein-Barr Virus (EBV) is a lymphotropic virus in humans that is closely associated with two malignancies, Burkitt""s lymphoma, and nasopharyngeal carcinoma, as well as a lymphoproliferative disorder, infectious mononucleosis. Also, in recent years, several EBV-associated proliferative syndromes and malignancies have been described in the profoundly immunocompromised host.
There are two main types of EBV carrying B lymphocyte lines, i.e., Burkitt""s Lymphoma derived (BL) and lymphoblastoid (normal lymphocyte) derived cell line (LCL). BL lines, which are derived from malignant cells in tumor biopsies, are monoclonal, usually aneuploid with a specific chromosomal translocation, bear a characteristic glycoprotein pattern, and are tumorigenic in nude mice. The LCL lines are derived from normal B cells, are polyclonal, have a normal diploid karyotype, a glycoprotein pattern similar to stimulated normal B cells, and do not grow when explanted subcutaneously into nude mice. LCLs, however, do grow in nude mice when inoculated intracerebrally, suggesting that immunological restriction is important in controlling outgrowth of EBV-carrying cells, even in a xenogeneic host. Reports of polyclonal outgrowths of karyotypically normal EBV-carrying cells in immunodeficient individuals confirm this observation (Houweling, A., P. J. van den Elsen, A. J. van der Eb [1980] Virology 105:537; Rassoulzadegan, M., A. Cowie, A. Carr, N. Glaichenhaus, R. Kamen, F. Cuzin [1982] Nature 300:713; Treisman, R., U. Novak, J. Favaloro, R. Kamen [1981] Nature 292:595; Giovanella, B., K. Nilsson, L. Zech, O. Yim, G. Klein, J. S. Stehlin [1979] Int. J. Cancer 24:103).
In general, tumor cells develop from normal cells by a multistage process. Two critical stages include (a) immortalization, i.e., the ability to divide perpetually without exogenously supplied mitogenic stimuli, and (b) acquisition of resistance to negative homeostatic signals that normally regulate growth. These stages may be associated with cytokines because regulation of proliferation and differentiation in most eukaryotic cells is accomplished by the interaction of specific cytokines with cell surface receptors. Receptor activation is followed by transmembrane signal transduction which leads to the generation of specific second messenger molecules. These receptor dependent events result in a defined series of cytoplasmic and/or nuclear changes leading to regulation of cellular activity.
Autonomous growth, as a result of transformation associated events, occurs in normal B cells transformed in vitro by EBV, and also in B cells derived from EBV positive and negative malignancies. EBV-transformed normal B lymphocytes divide continuously in culture without help from T cells or macrophages. Factor dependent autostimulatory growth for EBV-carrying B lymphocytes has now been reported by many groups. This secreted growth enhancing activity is specific for mature lymphoid cells. In addition, immortalized EBV-carrying B cells respond differently than normal B cells to certain cytokines, e.g., they proliferate in response to TGFxcex2 and IL-6. After EBV infection, B lymphocytes have an altered morphological appearance, produce immunoglobulin, and become independent of exogenous differentiation factors and resistant to saturation conditions in cell culture.
How lymphoid cells communicate with each other to affect cell growth, differentiation, and functional activities has been a major focus of investigation. The immune response to foreign antigens is dependent on the interactions of several different cell types, including macrophages, T, and B lymphocytes. The first described soluble growth factor of lymphoid origin, T cell growth factor (IL-2), was found in supernatants of lectin stimulated peripheral blood lymphocytes. Since the discovery of IL-2, various studies have described many additional growth factors and have begun to delineate the mechanisms controlling lymphocyte proliferation.
For the B lymphocyte, our understanding of the regulation of growth and differentiation has increased in complexity in the past few years. A plethora of factors, including BCGF (12 and 60 kD), IFNxcex3, TNFxcex1, lymphotoxin, TGFxcex2, IL-1xcex1, IL-1xcex2, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-15 and C3 fragments have been reported to modulate the growth of human and/or murine B lymphocytes in cell culture studies. These effects include growth augmentation (BCGF), differentiation (IL-6), and inhibition of proliferation (TGFxcex2). It appears that many of the T lymphocyte derived immunomodulatory molecules which direct B lymphocyte activities also regulate pleomorphic T cell functions. Some affect growth (IL-2), whereas others can also cause changes in cellular differentiation (IL-3). IL-2 functions as a direct growth factor for T lymphocytes, but other factors (IL-1) are described as comitogens or xe2x80x9chelperxe2x80x9d factors for cells stimulated by antigen or mitogen. IL-1, however, has recently been reported to function as a direct growth factor for one T cell sub-clone (Orencole, S. F., C. A. Dinarello [1989] Cytokine 1:14). For murine lymphocytes, two distinct CD4 expressing helper T cell subsets, Th1 and Th2, have been identified which differ in terms of factor response and production. Strictly similar subsets of distinct human T lymphocytes have not yet been described. Murine Th2 T cells secret IL-10, a 17 kD molecule which can inhibit Th1 cell activity. IL-10 has extensive homology with an uncharacterized EBV gene, BCRF1 (Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C. Sequin, P. S. Tuffnell, B. G. Barrell [1984] Nature 310:207).
Several growth factors have been proposed to have stimulatory activity for EBV transformed cells. These include BCGF (Ambrus, J. L., A. S. Fauci [1985] J. Clin. Invest. 75:732), IL-1 (Blazar, B. A., L. M. Sutton, M. Strome [1986] Canc. Immunol. 22:62), CD23 (Swendeman, S., D. A. Thorley-Lawson [1987] EMBO J. 6:1637), an unidentified lymphokine, called autocrine B Growth Factor, aBGF (Buck, J., U. Hammerling, M. K. Hoffmann, E. Levi, K. Welte [1987] J. Immunol. 138:2923), and, most recently, IL-6 (Muraguchi, A., H. Nishimoto, N. Kawamura, A. Hori, T. Kishimoto [1986] J. Immunol. 137:179). Thus far, none of these molecules has been shown to be universally present or absolutely required for growth of EBV positive lymphoblastoid cell lines. BCGFs of 25-30 kD and 60 kD, which are similar to T cell derived lymphokines, have been identified in supernatants from EBV-carrying cells. 60 kD BCGF has been purified to homogeneity and, although an activator of stimulated normal B cells, it is not produced by all EBV-carrying lines or even by all cells in individual secretor lines. BCGF production has also been reported for activated normal B lymphocytes (Muraguchi et al., supra).
Certain EBV-carrying cells have been reported to function as antigen presenting cells, contain IL-1 like activity in their supernatants, and express mRNA for IL-1. One laboratory reported the purification of a novel IL-1 from an EBV-carrying line (Bertoglio, J., J. Dosda, R. Stancou, E. Wollman, D. Fradelizi [1989] J. Mol. Cell Immunol. 4:139) but later revised their findings (Bertoglio, J., E. Wollman, A. Shaw, L. Rimsky, D. Fradelizi [1989] Lympho. Research 8:19). This same laboratory now reports that an IL-1-like activity (ADL) is elicited by a 12 kD protein produced by both EBV and HTLV-1 transformed cells (Wakasugi, H., N. Wakasugi, T. Trusz, Y. Tagaya, J. Yodoi [1989] J. Immunol. 142:2569; Tagaya, Y., Y. Maeda, A. Mitsui, N. Kondo, H. Matsui, J. Hamuro,, N. Brown, K. Arai, T. Yokota, H. Waksugi, J. Yodoi [1989] EMBO J. 8:757). Cloning of this protein, ADL, indicates it is a member of the human thioredoxin family with no direct relatedness to IL-1, although it may enhance IL-1 functions. Vigorous attempts by our laboratory using both Northern blotting and reverse transcription polymerase chain reaction (RT-PCR) demonstrate clearly that neither IL-1xcex1 nor IL-1xcex2 is expressed by these NAD-20 cells.
CD23, originally identified as a 45 kD differentiation antigen on EBV-infected cells, is expressed on all activated human B cells and macrophages. CD23 is identical to the low affinity Fc receptor for IgE (FceR11/CD23) (Defrance, T., J. P. Aubry, F. Rousset, B. Vandervliet, J. Y. Bonnefoy, N. Arai, Y. Takebe, T. Yokota, F. Lee, K. Arai, J. deVries, J. Banchereau [1987] J. Exp. Med. 165:1459). A soluble 25 kD form of CD23 is shed into cell supernatants. At present, there is much interest in CD23 because the latent EBV genes, EBNA 2 and LMP, appear to induce its expression in B lymphocytes (Wang, F., C. D. Gregory, M. Rowe, A. B. Rickinson, D. Wang, M. Birkenbach, H. Kikutani, T. Kishimoto, E. Kieff [1987] Proc. Natl. Acad. Sci. USA 84:3452). Some reports have suggested that it is a receptor for the low molecular weight BCGF (Gordon, J., A. Webb, G. R. Guy, L. Walker, M. Rowe [1986] Eur. J Immunol. 16:1627), and a shed form has been reported to function as an autocrine growth factor (Swendeman et al., supra). Conflicting reports, however, have also appeared. Recombinant shed FceR11/CD23 did not stimulate B cell proliferation, whereas it did bind IgE (Uchibayashi, N., H. Kikutani, E. L. Barsumian, R. Hauptmann, F.-J. Schneider, R. Schwendenwein, W. Sommergruber, W. Spevak, I. Maurer-Fogy, M. Suemura, T. Kishimoto [1989] J. Immunol. 142:3901). Highly purified shed CD23 from supernatants failed to stimulate B cell growth, and CD23 on the plasma membrane was not demonstrated as the receptor for the low molecular weight BCGF.
Acquisition of an autocrine growth cycle, whereby a cell both secretes and responds to endogenous growth stimulating factors, may be one means by which cancer cells achieve autonomy. Normal B lymphocytes transformed by EBV and malignant cell lines containing the EBV genome have been found to produce autostimulatory growth factors (Blazar, B. A., L. M. Sutton, M. Strome [1983] Can. Res. 43:4562).
The subject invention concerns the discovery and purification of a novel secreted cytokine from Mycoplasma. This protein has been named IL-X. IL-X, which can be isolated by size exclusion HPLC on TSK-SW-3000 columns, migrates at an apparent molecular weight of approximately 42 kD in SDS-polyacrylamide gels. Thus, this protein is referred to herein as the 42 kD IL-X protein, but it should be recognized that, due to the nature of SDS-PAGE analysis, the molecular weight of the protein may deviate slightly from the 42 kD value. IL-X stimulates proliferation of both EBV-carrying B lymphocytes and CON A activated normal T lymphocytes, suggesting a diverse role in control of lymphocyte proliferation. Growth of EBV-carrying lymphoblastoid cells is significantly reduced by antibody directed against either native IL-X or IL-X derived peptides (p-IL-X).
IL-X, either alone or in combination with other immunoregulatory molecules, can contribute to the establishment of long term lines of normal B cells in vitro. Such lines can also be used to generate monospecific human antibodies.
Severe pathology resulting from the uncontrolled proliferation of EBV-transformed B cells occurs in neoplasia and fatal infectious mononucleosis, as well as in situations of immunosuppression, immunodeficiency, and AIDS. Antibodies that neutralize IL-X activity, antisense DNA, or other antagonists of IL-X can be used to inhibit IL-X bioactivity or IL-X receptor function. For example, antibodies or other antagonists can be used to interrupt autocrine loops established in B cell neoplasia. Alternatively, exogenous replacement of IL-X can ameliorate certain B cell or T cell immunodeficiencies.
The subject invention also concerns isolated polynucleotide molecules which encode IL-X proteins.